1. Field of the Invention
The present invention relates generally to the field of tubulin polymerization inhibitors. More particularly, it concerns the use of 3-aroyl-2-aryl-benzo[b]thiophenes and analogues thereof as anti-tumor agents.
2. Description of Related Art
An aggressive chemotherapeutic strategy toward the treatment and maintenance of solid-tumor cancers continues to rely on the development of architecturally new and biologically more potent anti-tumor, anti-mitotic agents. A variety of clinically-promising compounds which demonstrate potent cytotoxic and anti-tumor activity are known to effect their primary mode of action through an efficient inhibition of tubulin polymerization (Gerwick et al.). This class of compounds undergoes"" an initial binding interaction to the ubiquitous protein tubulin which in turn arrests the ability of tubulin to polymerize into microtubules which are essential components for cell maintenance and cell division (Owellen et al.).
Currently the most recognized and clinically useful tubulin polymerization inhibitors for the treatment of cancer are vinblastine and vincristine (Lavielle, et al.). Additionally, the natural products rhizoxin (Nakada, et al., 1993a and 1993b; Boger et al.; Rao et al., 1992 and 1993; Kobayashi et al., 1992 and 1993) combretastin A-4 and A-2 (Lin et al.; Pettit, et al., 1982, 1985, and 1987) and taxol (Kingston et al.; Schiff et al; Swindell, et a, 1991; Parness, et al.) as well as certain synthetic analogues including the 2-styrylquinazolin-4(3H)-ones (SQO) (Jiang et al.) and highly oxygenated derivatives of cis- and trans-stilbene (Cushman et al.) and dihydrostilbene are all known to mediate their cytotoxic activity through a binding interaction with tubulin. The exact nature of this interaction remains unknown and most likely varies somewhat between the series of compounds.
Tubulin is a heterodimer of the globular xcex1 and xcex2 tubulin subunits. A number of photoaffiniity labeling reagents for tubulin have been developed and evaluated (Rao et al., 1992 and 1994; Chavan et al.; Sawada et al., 1991, 1993a and 1993b; Staretz et al.; Hahn et al; Wolff et al.; Floyd et al.; Safa et al.; Williams et al.). These reagents have identified three distinct small molecule binding sites on tubulin: the colchicine site, the vinblastine site and the maytansine/rhizoxin site. Additionally, a first generation rhizoxin-based photoaffinity labeling reagent has suggested binding to the Met-363-Lys-379 site on xcex2-tubulin (Sawada et al., 1993a), and a taxol-based reagent has been found to label the N-terminal 31 amino acid residues of xcex2-tubulin (Swindell et al, 1991 and 1994; Rao et al., 1994). Taxol itself is known to bind to polymerized microtubules, but not at distinct sites on the monomer subunits of tubulin (Kingston et al.; Schiff et al.; Swindell et al., 1991; Parness et al.).
The discovery of new antimitotic agents may result from the judicious combination of a molecular template which in appropriately substituted form (i.e. phenolic moieties, etc.) interacts with the estrogen receptor suitably modified with structural features deemed imperative for binding to the colchicine site on xcex2-tubulin (arylalkoxy groups, certain halogen substitutions, pseudo aryl ring stacking, etc.). The methoxy aryl functionality seems especially important for increased interaction at the colchicine binding site in certain analogs. (Shirai et al., D""Amato et al., Hamel, 1996). Recent studies have shown that certain estrogen receptor (ER) binding compounds as structurally modified congeners (2-methoxyestradiol, for example) interact with tubulin and inhibit tubulin polymerization. (D""Amato et al., Cushman et al., 1995, Hamel, et al., 1996, Cushman et al., 1997). Estradiol is, of course, perhaps the most important estrogen in humans, and it is intriguing and instructive that the addition of the methoxy aryl motif to this compound makes it interactive with tubulin. As a steroid, however, the use of 2-methoxyestradiol as an anti-cancer agent may lead to unwanted side effects.
Even before the discovery and realization that molecular templates (of traditionally estrogen receptor active compounds) suitably modified with alkoxyaryl or other groups deemed necessary for tubulin binding often result in the formation of new classes of inhibitors of tubulin polymerization, antiestrogens were developed to treat hormone-dependent cancers and a number of nonsteroidal agents were developed. Tamoxifen, for instance, has been widely used to treat estrogen-dependent metastatic mammary carcinoma (Mouridsen, et al.). The structure of trioxifene mesylate, a tetralin based compound which exhibits anti-tumor effects at the same or higher level as tamoxifen (Jones et al., 1979), includes a ketone moiety as part of its triarylethylene core, thereby overcoming the isomerization tendencies of the ethylene double bond of this class of compounds, assuring the stability of the molecule""s three-dimensional structure. Unfortunately, despite their antiestrogen properties, tamoxifen and the related triarylethylene derivatives retain some intrinsic estrogen agonist properties, reducing their ability to fully inhibit biological responses to exogenous or endogenous estrogens (Jones et al., 1984).
The benzo[b]thiophenes are another example of a class of compounds which often exhibit very high affinity for the estrogen receptor (Kym et al.; Pinney et al., 1991a and 1991b; WO 95/10513). The 2,3-diaryl substituted benzo[b]thiophenes greatly resemble the triarylethylene-based core structure of tamoxifen. The estrogenicity of the triarylethylene compounds has been shown to be substantially overcome in 3-aroyl-2-arylbenzo[b]thiophene compounds substituted at the 3-aroyl group with basic amine moieties (Jones et al., 1984). A prime example of this type of compound is LY117018 (U.S. Pat. No. 4,656,187). 3-aroyl-2-arylbenzo[b]thiophenes have also been found to be useful antifertility agents (U.S. Pat. No. 4,133,814) and as inhibitors for 5-lipoxygenase (U.S. Pat. No. 5,532,382).
The present invention provides benzo[b]thiophene-based inhibitors of tubulin polymerization, thereby providing novel anti-tumor compounds of increased cytotoxicity and fewer side effects. This is accomplished through the introduction of small alkoxy aryl substituents to the estrogenic benzo[b]thiophene skeleton or the skeleton of compounds similar to benzo[b]thiophene, such as indene, benzofuran, and indole. The tubulin polymerization inhibitors of this invention are illustrated by the structure: 
wherein
X is S, O, NH, or CH2,
R1-R4 are independently chosen from the group including H, OH and C1-C5 alkoxy,
Z is Cxe2x95x90O, CH2, C2H2, CHOH, or CHOCH3,
Y is a covalent bond, CH2, or CH2CH2 
Ar and Arxe2x80x2 are aryl moieties, chosen from the group consisting of phenyl and napthyl, wherein each aryl group is further substituted with at least one C1-C5 alkoxy group.
Preferably, the tubulin polymerization inhibitors of this invention will be of the above formula wherein X is S. The most preferred R group substitution pattern will be wherein R3 is OCH3 and R1, R2 and R4 are H. Z will preferably be Cxe2x95x90O, Y will preferably be a covalent bond, and Ar will preferably be 4-methoxyphenyl. The most preferred Arxe2x80x2 groups will be singly and multiply substituted phenyl groups containing para ethoxy or methoxy substituents. The most preferred tubulin polymerization inhibitor of this invention is 3-(3xe2x80x2,4xe2x80x2,5xe2x80x2-trimethoxybenzoyl)-2-(4xe2x80x2-methoxyphenyl)-6-methoxybenzo[b]-thiophene.
The term xe2x80x9cC1-C5 alkoxyxe2x80x9d as used herein contemplates both straight chain and branched chain alkyl radicals and therefore defines groups such as, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butyloxy, isobutyloxy, tert-butyloxy, sec-butyloxy, pentyloxy, isopentyloxy, t-pentyloxy, neopentyloxy, and the like. The preferred alkoxy groups are methoxy and ethoxy.
The novel compounds of this invention are of the structure: 
wherein
X is S, O, NH, or C2,
R1-R4 are independently chosen from the group including H, OH and C1-C5 alkoxy,
Z is Cxe2x95x90O, CH2, C2H2, CHOH, or CHOCH3,
Y is a covalent bond, CH2, or CH2CH2,
Ar and Arxe2x80x2 are aryl moieties, chosen from the group containing phenyl and napthyl, each aryl group substituted with at least one C1-C5 alkoxy group; wherein when Arxe2x80x2 is 3,4,5-trimethoxyphenyl or 4-methoxyphenyl, X is S, Z is Cxe2x95x90O, Y is a covalent bond, R3 is OCH3, R1, R2, and R4 are H, and Ar is a phenyl group that contains at least one methoxy substituent, then Ar must be substituted with a total of at least two alkoxy groups.
The preferred novel compounds of this invention will be those wherein X is S, Z is Cxe2x95x90O, R3 is methoxy and Ar is 4-methoxyphenyl. The preferred novel compounds of this invention include:
3-(2xe2x80x2,6xe2x80x2-dimethoxybenzoyl)-2-(4xe2x80x2-methoxyphenyl)-6-methoxybenzo[b]thiophene,
3-(3xe2x80x2,5xe2x80x2-dimethoxybenzoyl)-2-(4xe2x80x2-methoxyphenyl)-6-methoxybenzo[b]thiophene,
3-(3xe2x80x2,4xe2x80x2-dimethoxybenzoyl)-2-(4xe2x80x2-methoxyphenyl)-6-methoxybenzo[b]thiophene,
3-(4xe2x80x2-ethoxybenzoyl)-2-(4xe2x80x2-methoxyphenyl)-6-methoxybenzo[b]thiophene,
3-(3xe2x80x2,4xe2x80x2,5xe2x80x2-triethoxybenzoyl)-2-(4xe2x80x2-methoxyphenyl)-6-methoxybenzo[b]-thiophene, and
3-[3xe2x80x2-(3xe2x80x3,4xe2x80x3,5xe2x80x3-trimethoxyphenyl)propanoyl]-2-(4xe2x80x2-methoxyphenyl)-6-methoxybenzo[b]thiophene.
The most preferred novel compounds of this invention will be those wherein X is S, Z is Cxe2x95x90O, Ar is 4-methoxyphenyl, R3 is methoxy, and Arxe2x80x2 is a phenyl group substituted with an alkoxy group at the para position. The most preferred novel compounds of this invention include:
3-(3xe2x80x2,4xe2x80x2-dimethoxybenzoyl)-2-(4xe2x80x2-methoxyphenyl)-6-methoxybenzo[b]thiophene,
3-(4xe2x80x2-ethoxybenzoyl)-2-(4xe2x80x2-methoxyphenyl)-6-methoxybenzo[b]thiophene,
3-(3xe2x80x2,4xe2x80x2,5xe2x80x2-triethoxybenzoyl)-2-(4xe2x80x2-methoxyphenyl)-6-methoxybenzo[b]-thiophene, and
3-[3xe2x80x2-(3xe2x80x3,4xe2x80x3,5xe2x80x3-trimethoxyphenyl)propanoyl]-2-(4xe2x80x2-methoxyphenyl)-6-methoxybenzo[b]thiophene.
As a preferred embodiment of the invention, the tubulin polymerization inhibitors will be used as part of pharmacologically active compositions for treating leukemias, melanomas, and colon, lung, ovarian, CNS, and renal cancers, as well as other cancers. In the most preferred embodiment of this aspect of the invention, the tubulin polymerization inhibitors will be used to treat colon cancers.
As a further preferred embodiment, the tubulin polymerization inhibitors of this invention may be used to treat any disease for which tubulin polymerization plays a crucial role. In addition to anti-tumor activity, caused by lack of mitosis in cells in which tubulin polymerization is absent, the tubulin polymerization inhibitors of this invention would also be useful in treating diseases caused by flagellated parasites, for whom tubulin polymerization is crucial to movement. In particular, the tubulin polymerization inhibitors of this invention will be useful in treating Chagas"" disease or diseases caused by the parasite Leishmania.
The present invention also includes a compound of the structure 
where R1 is H or CH3O; R2 is H, CH3O or C2H5O; R3 is CH3O or C2H5O; R4, R5, R7 and R8 are independently H, CH3O, C2H5O, or F; R6 is H, CH3O, C2H5O, OH, F or N(CH3)2; and X is 
The present invention also includes a compound of the structure 
where X is S or Sxe2x95x90O.
Also included is a compound of the structure 
as well as a compound of the structure 
where X is CHOH or Cxe2x95x90O. A compound of the following structure is also included in the present invention 
where R1 and R2 are independently CH3O, NO2, NH2 or N3 and CH3O; and C2H2 is in the E or Z configuration; with the proviso that one of R1 and R2 is CH3O and the other is NO2, NH2 or N3.
One preferred embodiment is where C2H2 is E C2H2, R1 is CH3O and R2 is NH2 in the immediately prior structure. Another embodiment is where C2H2 is E C2H2, R1 is NH2 and R2 is CH3O in the immediately prior structure. Yet another embodiment is where C2H2 is Z C2H5, R1 is CH3O and R2 is NH2 in the immediately prior structure. Another embodiment is where C2H2 is Z C2H2, R1 is NH2 and R2 is CH3O in the immediately prior structure. Another embodiment is where C2H2 is E C2H2, R1 is CH3O and R2 is NO2 in the immediately prior structure. In another embodiment C2H2 is E C2H2, R1 is NO2 and R2 is CH3O in the immediately prior structure.
One preferred embodiment of the present invention is where C2H2 is Z C2H5, R1 is CH3O and R2 is NO2 in the immediately prior structure.
One embodiment is where C2H2 is Z C2H2, R1 is NO2 and R2 is CH3O in the immediately prior structure.
Yet another embodiment is where C2H2 is E C2H2, R1 is CH3O and R2 is N3 in the immediately prior structure. A preferred embodiment also is where C2H2 is E C2H2, R1 is N3 and R2 is CH3O in the immediately prior structure. Also preferred is the compound where C2H2 is Z C2H5, R1 is CH3O and R2 is N3 in the immediately prior structure. Another embodiment described is where C2H2 is Z C2H2, R1 is N3 and R2 is CH3O in the immediately prior structure.